PROJECT SUMMARY/ABSTRACT Chagas disease is caused by the parasite Trypanosoma cruzi, which chronically infects cardiac myocytes, leading to significant cardiac pathology over the lifetime of the host. There is a fundamental gap in the understanding of T. cruzi?s affinity for the human myocardium. Studies in non-cardiac cells and rodents have implicated lipid metabolism as an important factor in the intracellular maintenance of T. cruzi, but data specific to cardiac myocyte metabolism and the lipid products resulting from the parasite-host interaction are lacking. Shifting the focus to the role of cardiac tissue is crucial for understanding the natural history of human T. cruzi infection. The objective of this research is to identify metabolic factors specific to human cardiac myocytes that are necessary for T. cruzi infection. The central hypothesis is that specific cardiac lipids or lipid metabolites are essential nutrient sources for intracellular replication and maintenance of T. cruzi, leading to this parasite?s tissue affinity and subsequent cardiac pathology. The long-term goal is to use this understanding of host- parasite interaction to develop precision cardiac diagnostics and improve therapeutic options for this insidious and debilitating infection. Understanding the role of human myocardial metabolism in the regulation of T. cruzi infection will be tested with two specific aims: 1) evaluation of lipidomic perturbances in T. cruzi-infected cardiac myocytes and 2) determining human cardiac metabolic regulators of T. cruzi infection. Aim 1 will employ a mass spectrometry-based lipidomic workflow, established by the applicant, to quantify hundreds of biologically relevant lipids during a 6-day T. cruzi infection cycle in induced pluripotent stem cell-derived cardiac myocytes (iPS-CMs). Significantly dysregulated lipids in culture media and cell lysates will be validated with individual stable isotope-labeled lipid standards to determine incorporation of heavy-labeled components into key lipid metabolism pathways in T. cruzi, harvested post host cell lysis. Aim 2 will employ a first-of-its-kind CRISPR interreference (CRISPRi) screen of human metabolism-related genes in cardiac myocytes to evaluate physiologically relevant host factors affecting T. cruzi replication and maintenance. These experiments will utilize iPS-CMs harboring CRISPRi machinery transduced with a focused guide RNA library to knock down human metabolic genes. After infection with green fluorescent protein (GFP)-tagged T. cruzi, we will sequence iPS-CM populations harboring high, normal, and low parasite burdens. These experiments will reveal the relevant gene knockdown targets in each replication phenotype to provide insight into key cardiac regulators of infection. This approach is innovative because it combines state-of-the-art technologies in lipidomics and CRISPR-based screening to investigate the interface of host-pathogen metabolism in a physiologically relevant cardiac model. The proposed research is significant because it will provide biological evidence for specific aspects of lipid metabolism in the cardiac tropism of T. cruzi, which will provide a foundation on which to develop precision cardiac diagnostics and novel, evidence-based therapies.